The evolution of regeneration: Adaptive or inherent?

J. theor. Biol. (1992) 159, 241-260

The Evolution of Regeneration: Adaptive or Inherent? RICHARD J. Goss Division of Biology and Medicine, Brown University, Providence, RI 02912, U.S.A. (Received on 10 April 1992, Accepted in revised form on 12 June 1992) If regeneration were adaptive, it would have arisen autonomously by natural selection from non-regenerative antecedents. Unless each episode coincidentally reinvented the same method of regeneration independently, one would expect the various lineages to differ basically from each other, which they do not. On the other hand, if regeneration were inherent to metazoan life, a derivative of embryogenesis, its various expressions should be as much like each other as they resemble the develolrment of embryonic appendage buds, which they do. It follows that the uneven distribution of regeneration must have been due to its extinction here and there, not as a negative adaptation by natural selection but as a pleiotropic epiphenomenon linked to more useful adaptations with which it was incompatible. In vertebrate evolution, these adaptations have included the transition from aquatic to terrestrial habitats and the modification of poikilothermic to homeothermic metabolism. The former advance rendered the regeneration of weight-bearing limbs impractical; the latter favored rapid wound healing and scar formation which effectively precluded blastema formation. If the latent capacity for regeneration persists in non-regenerative appendages, as would seem to be the case, then the restoration of its overt expression should be possible if the mechanisms of its inhibition could be discovered and eventually rendered ineffectual. "There is somethingfascinating about science. One gets such wholesale returns of conjecture out of such a trifling investment of fact". Mark Twain, Life on the Mississippi

1. Historical In the 1890s there erupted an unseemly debate between the great German biologist, August Weismann, then in his sixties, and the not yet renowned Thomas Hunt Morgan, barely into his thirties. The publication of Weismann's opus, "The GermPlasm", in 1893 included a chapter on regeneration in which its adaptive nature had been proposed. "The capacity for regeneration", he wrote (p. 114), "is not a primary quality of the organism, but . . . is a phenomenon of adaptation". "The power of regeneration", he continued (p. 118), "is graduated according to the need . . . . The degree in which it is present is mainly in proportion to the liability of the part to injury". The skeptical Morgan (1898"287) wrote, "I cannot believe that the chapter on regeneration in The Germ-Plasm will convince anyone that the phenomena are in anyway explained. For myself I fail to see by what nice mechanism the power of regeneration is graduated according to the need of regeneration of 241

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each part. The 'Natur-philosophie' seems not yet dead". The next year, Weismann (1899: 317) replied: "Morgan casts a glance at my theory of regeneration as an adaptation-phenomenon, and not exactly a friendly glance either; for it seems to be the fashion nowadays among the younger investigators to look down with a certain lofty disdain upon so-called 'explanations' which are based upon the selectionhypothesis". When Morgan cast aspersions at the long since discredited "Naturphiiosophie", he was attempting to disparage Weismann's theory by lumping it with the romantic and transcendental mysticism so popular in the previous century. Not to be put down, Weismann (1899: 317) replied to Morgan's statement about the demise of Natur-philosophie as follows: "It is to be hoped that it is not, and that it never will be dead, for progress in our knowledge will always depend upon the philosophical treatment of known facts, since it is only in this way that we can set up new goals to guide our observations . . . . " Transcending the ad hominem dialectic between Morgan and Weismann is the perennial problem of whether regeneration arose once or many times in the course of metazoan evolution. Weismann was simply reiterating the earlier opinions of de Rraumur (1742) and Bonnet (1745) that regeneration of lost parts was adaptive, a viewpoint upheld many years later by Needham (1961a, b), Scadding (1977), and Reichman (1984). Morgan's interpretation, on the other hand, was echoed by Korschelt (1927) who believed that regeneration was an "Ursprungserscheinung", a primordial phenomenon of life. In 1943, Huxley wrote (p. 418) that "Regeneration is to-day universally looked upon as one aspect of an inherent quality of life, and the chief problem set by it to biology is not how to account for its presence in the lower forms, but how to explain its restriction and absence in higher types". The problem remains unsettled to this day. The question is far from academic. It has profound significance with respect to our hopes eventually to induce regeneration where it does not naturally occur, as for example in our own arms and legs. If epimorphic regeneration has arisen repeatedly and separately in various animals and their appendages, then each example would be uniquely different from all others. In which case, the chances of reinventing it on our own would seem to be very slim indeed. But if it were a primeval attribute of metazoan life, the phenotype might have been eliminated here and there during the course of evolution, but its genotype could have persisted. It would seem reasonable to expect that the basic capacity to regenerate might lie latent in non-regenerative structures, available to be released if only we knew what blocked it and how to reverse the blockade. The latter possibility is greatly desired because it would make the task of restoring regeneration easier than having to recreate it from scratch. August Weismann goes down in history for emphasizing the continuum of all life, perpetuated through countless generations by way of the germ cells. Each egg, or zygote, would develop into an organism whose function it was to reproduce more of its kind before dying. The demise of the transitory soma was thus transcended by the survival of its germ cells in subsequent cycles of reproduction ad infinitum. The units of heredity, unknown in the era before the discovery of genes and the rediscovery of Mendel, were carried in the immortal germ cells, and were responsible in ways as yet to be disclosed for the embryonic development of the somatic parts of the

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body. Different types of somatic cells presumably represented their own fractions of the overall inheritance. Such a perspective worked fine until the problem of regeneration arose. If a worm could grow back substantial parts of its body after bisection, or could even reproduce vegetatively by developing copies of itself spontaneously from intact segments of its body, the role of germ cells as exclusive vehicles of heredity was open to reinterpretation. The implication was that each somatic cell was endowed with as complete a set of genetic determinants as the germ cells, except that their expression was more limited. Only when induced to regenerate were their latent potentialities realized. It was such considerations that led Weismann to speculate on the evolution of regeneration (and to run the risk of being accused of reviving Natur-philosophie). 2. Adaptation and Natural Selection There are two kinds of adaptation. Physiological adaptation represents those functional changes by which an organism remains attuned to its environment. Such adjustments may be metabolic, developmental, or behavioral, but in any case are compensations for the vicissitudes of the environment. "The organism changes geometrically", wrote Brody (1945: 580), "so as to remain the same physiologically". The other kind of adaptation is genetic. It is a modification that promotes an organism's ability to survive in its environment and to reproduce its kind. Genetic adaptations arise by natural selection as advantageous genes are more successfully perpetuated than disadvantageous ones. If developmental phenomena are conceded to be physiological processes, then those which occur in response to environmental stimuli may be classified as physiological adaptations. Appendage regeneration following amputation, wound healing in response to injury, compensatory growth (or atrophy) due to use (or disuse), and perhaps even physiological turnover if this is a reaction to the finite lifespans of cells and half lives of molecules, may all be regarded as proximate mechanisms by which normal morphology is restored despite disruptions. The genetic dimension of these various reparative processes resides in their distribution in the animal kingdom, or at least amongst the metazoans. The possibility that Weismann suggested was that regeneration might be adaptive, and therefore would have arisen through natural selection. It may be no coincidence that he did not also imply that wound healing, compensatory growth, and turnover, were adaptive. The reason he did not include these other processes was probably that they are universally distributed. Virtually all organs and organisms are endowed with these capacities, as not being capable of repairing injuries, of adjusting organ size to functional demands, or turning over molecules and cells would be incompatible with survival. These are obligatory processes, and as such they are ubiquitous. Anything that is ubiquitous must by definition be exempt from natural selection. Selection operates on alternatives, and there are no viable alternatives to these vitally essential phenomena. Regeneration, however, is unique in that it is the only developmental phenomenon that is not ubiquitous. It is more an optional luxury than a vital necessity. It is

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convenient to be able to grow back missing parts, but it is not always a matter of life or death. Yet is is difficult to imagine an instance of regeneration that would not be an advantage to an organism. Surely the restoration of lost parts would enhance an animal's ability to survive and reproduce. It seemed only logical to Weismann, therefore, that there ought to be a selective advantage favoring the adoption of such an obviously beneficial attribute. Clearly, animals that could grow back their amputated legs would on average produce more progeny than those crippled with unregenerated stumps. Weismann went on to propose that natural selection for regenerative ability would be an adaptation to the liability to injury. The higher the incidence of amputation, the greater the selective pressure to replace the lost part. Liability to injury is difficult, but not impossible, to quantify. The actual frequency of amputation can be measured in a population, and it is probably safe to assume that such data would be commensurate with exposure to potential contingencies. The highest incidences of loss and regeneration have been reported for appendages capable of autotomy. King (1898, 1900) found that 10.7% of 1914 starfish (Asterias oulgaris) had lost one or more arms. Morgan (1901) counted 21 out of 188 (11%) hermit crabs (Eupagurus longicarpus) that had missing legs. Werner (1968) reported up to 18-70% loss and/or regeneration in lizard tails, while Parker (1972) and Vitt et al. (1977) recorded frequencies up to 74%. Even among appendages not endowed with the capacity for autonomy, in which the number of amputations is often too low to count accurately, the need for regeneration may still be significant on a lifelong basis. For example, if only 1% of a population is observed to have lost a limb or to be in the process of regenerating one at a given time, but if the time required to complete regeneration is 5% of the animals' lifespans, then an average of 20% of the population would be expected to benefit from the capacity to regenerate during their lives. It is not known how high an incidence of loss is necessary to tip the selective scales in favor of regeneration, but in view of the amplification that accrues in longer lived animals it is hardly surprising that regenerative abilities are so widespread among cold blooded forms that sometimes exhibit surprising longevities (Comfort, 1964). As Needham (1961b) has written, "Successful regeneration, even if called for in only a small percentage of individuals, provided it enables them to make a further small contribution to the size of the next generation may make just the difference between an assured survival and an inevitable extinction for the species". In casting about for the ideal system in which to test Weismann's liability to injury hypothesis, both Morgan and Needham focused on the hermit crab. In one and the same animal, there were anterior legs (pereiopods) exposed to the external environment and vulnerable to injury, and abdominal appendages that ought seldom to be lost because they are protected inside the shell. If Weissmann was right, the anterior appendages should be lost more frequently than the abdominal ones, and they should therefore regenerate better. Morgan (1898) showed that 10% of the anterior appendages had been lost in 288 specimens examined, while only 4% of the abdominal legs were either amputated, absent, or smaller than normal, in 100 crabs. When tested for regenerative ability, however, all appendages proved capable, even the thoracic

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legs and uropods which had not been found to have been lost in nature. "In regard to the problem of the frequency of injury of a part and its capability to regenerate", Morgan (1898: 299) wrote, "no such relation is found to exist". His interpretation, however, was considered misleading by Needham (1961a) who repeated some of Morgan's studies and obtained similar results. Among 82 hermit crabs examined, 9-8% were regenerating anterior legs while only 3.7% were replacing abdominal ones. Although Morgan had emphasized that all appendages could regenerate despite their different susceptibilities to injury, Needham was more impressed with the differential regenerative frequencies that were actually correlated with liability to injury. He pointed out that Morgan's data showed that anterior legs grow back in 83% of the cases, while abdominal legs regenerated only 21% of the time. These findings seemed to confirm rather than confute Weismann's hypothesis that regeneration might be related to liability to injury. "Morgan tended to minimize quantitative difference in ability", wrote Needham (1961a), "and to magnify differences in susceptibility to loss". If regeneration arose by natural selection as Weismann contended, this would be consistent with its uneven distribution in the animal kingdom. Although natural selection is commonly associated with the evolution of structures adapted for particular circumstances, in the case of regeneration it would be selection for a process. The problem with this possibility is that the process is a very complex one. It includes epidermal wound healing, dedifferentiation, blastema formation, and morphogeuesis. These events at the cell and tissue levels are made possible by even more numerous reactions at the molecular level, each of which presumably depends upon specific gene derepressions. If it is assumed that regeneration might have arisen de n o o o by natural selection, a large constellation of mutations would have had to occur simultaneously in order for all of the basic chemical reactions required for regeneration to be activated at once. One cannot resort to arguments that regeneration could have evolved piecemeal, gradually building up step by step upon itself, for if it were to be subject to positive selective pressure, regeneration would have to be an all-ornone event. Natural selection generally operates on single mutations at a time, so if it were to be responsible for the occasional evolution of regenerative ability, all of the many subsidiary processes upon which regeneration depends would have to pre-exist. Such a scenario would be obtained if regeneration were to recapitulate ontogeny. Indeed, it is remarkable how closely regeneration does resemble embryogenesis. The same sequence of events is played out in the development of the blastema as occurs in the original development of the corresponding limb bud or tail bud in the embryo. This possibility would be compatible with the fact that regenerative processes in general all operate in very much the same way with only minor variations according to the differences between the different appendages capable of regrowth. According to this interpretation, regeneration would utilize the same sequence of genes operating in original embryogenesis (Barr, 1964). Inasmuch as nature is conservative, this would seem more plausible than evolving completely separate mechanisms for regeneration each time it has been needed during phylogeny. In point of fact, evolution proceeds not by engineering but by tinkering, as Jacob (1977) has .

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SO astutely noted. When a process evolves, it does so not by creating new genes augmenting the pre-existing genome, but by altering those already present. Evolution is thus constrained by its own genetic heritage, unable to "design" new structures or processes except by awaiting random mutations that may on rare occasions be adaptive. Notwithstanding the aforementioned similarities between regeneration and embryogenesis, Stocum (1991) has called attention to some interesting differences. In addition to the acquired dependence upon nerves exhibited by many (but not all) regenerating systems, there are several antigens present either in the wound epidermis or in the underlying mesenchymal cells of the regenerating limb blastema that are not represented in the embryonic limb bud. Also, the reactions of blastemas vis-d-vis limb buds to the effects of retinoic acid are quite different. Stocum concludes "that limb development and regeneration are distinct developmental processes". However, it would seem prudent to reserve judgment on this basic question pending more detailed comparisons between the two. Regeneration and embryogenesis could have been originally alike, for example, but in subsequent evolution either or both might have been modified in response to selective adaptations superimposed upon a fundamental process. If regeneration is adaptive, it evolved by natural selection from non-regenerative antecedents. As acquired traits, the many different examples of regeneration would have been polyphyletic in their origins, each one arising (and perhaps disappearing) independently. Wherever the incidence of amputation were high enough, selection would favor retention of any mutants suddenly endowed with regenerative abilities. Non-regenerative structures would presumably represent those not exposed to sufficiently life-threatening conditions to have been lost often enough to promote selection. As a contingent phenomenon, the capacity for regeneration cannot be expressed in the absence of amputation. Latent capacities are selectively neutral unless tested. The purported adaptive nature of regeneration presupposes that ancestral appendages were totally incapable of regeneration. Whenever the incidence of amputation exceeded a certain threshold the stage would be set for a regenerative mutation to occur and for natural selection to promote its spread throughout the population. What this threshold might be is open to speculation. A frequency of injury leading otherwise to the extinction of the species would obviously suffice, for only by the intervention of regeneration could such a fate be avoided. Amputations so infrequent as to have a negligible effect on the population as a whole would presumably be too trivial to benefit from the acquisition of regenerative ability. Between these two extremes, however, individuals endowed with the capacity to replace lost parts would survive and reproduce better than others, eventually diluting out non-regenerative congeners in the gene pool. After all, regenerative structures are accidentally amputated more often than non-regenerative ones, a tautology that is not coincidental. Morgan took issue with Weismann's hypothesis that regeneration was adaptive in virtue of its selective advantage in the course of evolution. This may have been due in part to his disinclination to believe that the basic mechanism of evolution could be explained in terms of natural selection. He was by nature an ardent experimentalist

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in the Entwicklungsmechanik tradition (Allen, 1978) who was skeptical of theoretical explanations that could not be tested, including phylogenetic ones. Perhaps he read into Weismann's ideas forbidden teleologic overtones whereby liability to injury might supposedly have conferred a creative dimension on natural selection. Natural selection, in Morgan's view, operated strictly by the process of elimination. Morgan's skepticism persisted throughout the rest of his life. As late as 1932 (pp. 130-131), he wrote that "the implication in the theory of natural selection, that by selecting the more extreme individuals of the population, the next generation will be moved further in the same direction, is now known to be wrong". " . . . even without natural selection", he added, "evolution might have taken place". With respect to regeneration, Morgan (1901:107) quite aptly pointed out that, "If, therefore, the animal can exist through the long interval that must elapse before the lost part regenerates, we cannot assume that the presence of the part is of vital importance to the animal, and hence its power to regenerate could scarcely be described as the result of a 'battle for existence' and without this principle 'natural selection' is powerless to bring about its supposed result". Further, he suggested that animals capable of avoiding injury in the first place were more adapted to their environment than others that might have to regenerate as a result of injury. "It is assumed", he wrote (Morgan, 1901: 109), "that those individuals that regenerate better than those that do not, survive, or at least have more descendants; but it should not be overlooked that the individuals that are not i n j u r e d . . , are in even a better position that those that have been injured and have only incompletely regenerated". He also contended that occasional regenerative forms, would be swamped out by non-regenerative ones by interbreeding, especially if the latter avoid injuries more successfully. Actually, Weismann apparently did not interpret his theory of adaptive regeneration to mean that each isolated instance of regeneration arose de nooo. He recognized that wherever regeneration occurred, it would have descended from regenerative ancestors. "Hermit-crabs have certainly possessed the power of regeneration 'from the beginning', but may they not have inherited it from their ancestors, the longtailed forms, which possess it to this day and have need of it for all their appendages since all are liable to injury? And cannot, nay, must not, these in their turn have inherited it from their ancestors, the sessile-eyed crustaceans, and so on through the whole crustacean-pedigree back to the unknown annelid-like ancestors of the class?... We know that the lower worms have quite as high a regenerative power, extending to all their parts, as the lower Coelentera or polypes for instance. It seems almost as if Morgan ascribed to me the view that the capacity for regeneration must be built up anew for each species--must be inscribed so to speak on a tabula rasa" (Weismann, 1899:311). These comments clearly bridge the gap between Morgan and Weismann, although neither would have conceded it. Weismann refused to admit to any general power of regeneration, but he was nonetheless unable to pinpoint just where in phylogeny regeneration began, preferring to view the role of natural selection as one of modifying regeneration already established. Weismann's hypothesis, therefore, was not so radical as Morgan had interpreted it. Inasmuch as he could not cite examples of non-regenerative structures evolving

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into regenerative ones, Weismann implied by default that regeneration must have prevailed "from the beginning". This actually concurred with the view proposed by Morgan (1901) and later by Korschelt (1927), that regeneration was a primitive character inherent in the earliest metazoans. There was no real need to explain the occurrence of regeneration by natural selection impelled by liability to injury. If it was monophyletic, its absence here and there in the animal kingdom could mean only one thing: that it had been eliminated in certain animals whose ancestors had once been endowed with regenerative powers.

3. Implications of Implicit Regeneration If in fact regeneration is a primeval quality of life, there follow several important consequences. One is that regeneration and embryogenesis are basically the same phenomenon. The hypothetical first metazoan may have multiplied by binary fission the way its protozoan ancestors did, a kind of vegetative reproduction to augment the sexual alternative. Such a process would depend on the reutilization of genes operating in sexually produced zygotes and embryos. Indeed, the very essence of regeneration itself is the reactivation of genes that program development in cells other than the gametes. This potential, which resides in all nucleated cells, is one which must be carefully regulated. Otherwise the organism stands in danger of all of its cells opting for an independent existence by initiating their own embryogenesis. This "somatic parthenogenesis" would be deleterious to the survival of the parental organism, but would have the advantage of rapid amplification of the population, albeit by clonal progeny produced without benefit of genetic recombinations. In this perspective, it is not so remarkable that vegetative propagation exists at all, but that it occurs as infrequently as it does. It would seem not to be a major innovation to adapt vegetative propagation, as it occurs spontaneously in some worms, for example, to the regeneration of accidentally bisected organisms. In principle, the same mechanisms would also be expected to operate in appendage regeneration in higher animals that are incapable of surviving bodily transection. Such examples of "adult embryogenesis" can thus be traced back to conventional embryonic development as it occurs in the zygote. This interpretation is consistent with the apparent fact that the same genes are used for regeneration as were responsible for enbryogenesis (Barr, 1964). It follows that various forms of regeneration should as closely resemble each other as they do embryogenesis. Yet they are not identical, for the many body parts capable of regeneration differ from one another. Also, regeneration takes place in an adult organism whose morphology and physiology differ drastically from its prenatal precursor. Although embryonic and regenerative development are basically the same, the latter has had to adapt to the adult milieu by including epidermal wound healing, dedifferentiation, blastema formation, and even dependence on innervation and hormones, in its developmental repertoire. It is no coincidence, however, that once a blastema has formed its subsequent development unfolds in much the same way as did that of the embryonic bud. Inductive interactions between ectoderm and mesoderm are found in both phenomena. Apical proliferation is typical of embryonic

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mesenchyme as well as blastema cells. Differentiation is initiated proximally and proceeds distally in both cases. The histogenesis of component tissues in the regenerate recapitulates that in the embryo. Although there are many ways one could imagine to develop a replacement of a lost structure, nature does it the economical way by using pre-existing mechanisms encoded in the genes. Another corollary of the monophyletic theory of regeneration is that all regenerative phenomena would have evolved in an unbroken line of descent from regenerative antecedents. This proposition fits the existing phylogenetic pattern of regeneration in so far as it is known. Wherever a structure can grow back missing parts, so also should the homolog from which it evolved. Amphibian limbs are believed to have evolved from the paired, lobed fins of ancestral Crossopterygians (Coates, 1991), so one might predict that such fins might regenerate in the Coelacanth, Latimeria, if these fishes were available for experimentation. Lizard tails evolved from those of their amphibian progenitors, which in turn can be traced back to the fishes. Unhappily, it is not possible to experiment on fossils to find out whether or not they could regenerate their appendages or tails. Although fortunate accidents of nature may occur [as in a fossil tail regenerate in a Carboniferous microsaurian amphibian described by Carroll & Baird (1968)], the cartilaginous nature of skeletal regenerates usually militates against their preservation in the paleontological record. It is noteworthy that when a given structure can regenerate, its serial homologs can do likewise. If a forelimb regenerates, so can its hindlimb. If the paired fins of a fish regenerate, the unpaired fins (dorsal, caudal, anal) can also regenerate. The same principle applies to the gills of fishes and amphibians, the diverse appendages of crustaceans, the arms of cephalopods, and the parapodia of polychaete anndids. Homologous structures come endowed with homologous regenerative abilities, regardless of their respective liabilities to injury. Just as the sheltered abdominal legs of the hermit crab can regenerate, so also can the forelimbs of anuran tadpoles that develop within the protected confines of the gill chamber. Although homologous structures, be they serial or phylogenetic, can regenerate, what about appendages that evolve independently without ancestral homologs? If regenerative ability is not to be acquired in a previously non-regenerative appendage, then newly evolved appendages must perforce possess such a capacity from the outset. The catfish, for example, evolved taste barbels around its mouth, appendages for which there are no counterparts in lower forms. Their ability to regenerate is well documented (Kamrin & Singer, 1953). Other fishes are adorned with fleshy protuberances which serve as camouflage, but the ability of tbese sometimes elaborate decorations to grow back seems not to have been investigated. Nor has the lure of the angler fish been tested for its regenerative ability, although its obvious liability to injury (and its homologous relationship to dorsal fin rays) would seem to suggest such a probability. Among reptiles, Jackson's chameleon grows three horns in the male, excrescences which remain to be explored for their potential capacity to be replaced after amputation. One wonders if Triceratops might have regrown its horns broken in combat. Certain birds possess combs and wattles, ornaments that apparently are not replaced following loss or injury. Various mammalian structures, such as the giraffe's horns, the moose's bell, the panda's thumb, and the elephant's trunk

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are not known to regenerate. The external ears of mammals do not regrow if amputated but in some forms (e.g. rabbits, bats, cats) they regenerate centripetally to fill in full thickness holes punched through them (Goss, 1980). Bat wings can also repair holes, but the comparable capacity in flying squirrels has not been examined. Neither the tongue, penis, nor foreskin can regenerate. Female breasts do not grow back, but there is some evidence for nipple regeneration in guinea-pigs, rabbits and rats (Markelova, 1952). The cornified horns of various ungulates do not regenerate after loss, but the antlers of deer, of course, are famous for their spontaneous regrowth every year after the old ones drop off (Goss, 1983). Finally, the star-nosed mole (Condylura cristata) comes to mind as a mammal which has recently evolved new appendages. Its 22 fleshy protuberances that surround the snout would seem to be vulnerable to injury, whatever their functions might be, yet they have not been put to the test to determine whether or not they might regenerate. Clearly, there are many cases of newly evolved appendages that are known not to regenerate, but there are others that do regenerate and still others whose capacities are unknown. Hypothetically, the more recently such structures may have evolved, the greater the probability that they might still retain a capability for regrowth. Whether they do or not depends on what factors might be responsible for the widespread loss of regeneration, especially in the higher vertebrates. 4. Loss of Regeneration Assuming regeneration to be implicit, but being well aware of its heterogeneous distribution within the animal kingdom, its disappearance from otherwise regenerative forms requires an explanation. To turn Weismann's argument around, one could suppose that natural selection might have favored the loss of regeneration in cases in which liability to injury fell below a critical threshold. It seems true that non-regenerative structures are very rarely amputated, but is this sufficient to bring about the loss of an unused ability? Can natural selection be negative? In reply to the latter question, the answer would seem to be affirmative. Evolution is replete with examples of structures that have disappeared or been reduced in size. The posterior appendages of cetaceans have been lost, presumably by selection against structures inimical to the need for a streamlined body. The wings of flightless birds have been reduced through disuse, and avian teeth have been eliminated altogether. Many cavernicolous animals conserve energy by doing away with eyes and pigment cells rendered unnecessary in total darkness. The neotenous axolotl has abandoned metamorphosis along with the amphibious life style of its ancestors. Granted that natural selection can work both ways, and that it can abolish maladaptive structures that are overtly deleterious, or not economical in the metabolic balance of the body, or simply neutral, it must be asked if a process as obviously important as regeneration could actually be selected out. It should if regeneration were disadvantageous. For example, the process of regrowth consumes energy, a trait that could prove fatal under conditions of inanition. Yet in its infinite wisdom, nature has not ordered its priorities by preventing regeneration of an appendage even when it might cost the life of an animal starving to death. Even though regeneration

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has been lost in higher vertebrates smart enough to anticipate danger and elude it (Elder, 1979), there is reason to believe that the latent capacity has persisted, as discussed below. There seems to have been a tendency in vertebrate evolution to conserve regeneration, either overt or covert, whether it is disadvantageous or unnecessary.

5. Pleiotropism There are two conditions under which regeneration does not take place. One is when a structure can regenerate but is never amputated. The other is when a structure is amputated but cannot regenerate. It is questionable whether or not the former circumstance is ever obtained; and, if it did, regenerative ability would be irrelevant. Why the latter condition is so prevalent among higher vertebrates is challenging to explain. What could be the reason for the unaccountable absence of regeneration where it might otherwise be expected to occur? It is doubtful that regeneration per se has been specifically selected against. Its absence is not an adaptation that would promote survival and propagation. Therefore, the lack of regenerative ability is probably an epiphenomenon linked (pleiotropically?) to other attributes with which it is incompatible. In vertebrates, these relate to the two most revolutionary advances that have occurred in evolution, the transition from aquatic to terrestrial habitats, and the change from cold blooded to warm blooded physiology. These two events could only have happened in the sequence listed. Aquatic animals could not become warm blooded owing to the unfavorable specific heat of water. Once they became terrestrial, in an atmosphere conducive to homeothermy, then they could secondarily return to the water with adequate insulation. Nevertheless, both of these transformations have been antagonistic to regeneration, each in its own way. The significant difference between the aquatic and terrestrial existence is not so much whether oxygen is extracted from water or air, nor that one is wet and the other dry, but that water has greater buoyancy than air. Most of the appendages of fishes and aquatic amphibians are not weight-bearing, in contrast to the walking legs of adult anurans, reptiles, birds and mammals. This correlation matches the capacities of such appendages to regenerate or not. Teleost fins and salamander legs regenerate nicely, while the limbs of reptiles, birds and mammals do not. Bottom-dwelling fishes regenerate their pectoral fins less well than others (Wagner & Misof, 1992), perhaps because they are in direct contact with a solid substrate. Presumably their delicate blastemas would be subject to injury and therefore incapable of developing normally. If this reasoning is correct, one would predict that the paired fins of mud skippers should also be non-regenerative. Scadding (1977, 1978) surveyed a variety of adult amphibians for their regenerative abilities. Among the salamanders he found that some of the aquatic forms (Amphiuma, Necturus, Siren) do not regenerate, while others do (Notophthalmus). Terrestrial urodeles also differ, Plethodon, Eurycea and some Ambystoma being able to regenerate, while others (some Ambystoma, Salamandra) cannot. These discrepancies may

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perhaps be explained by size differences. Scadding (1977) noted that small species regenerate well while large ones do not. Smaller terrestrial salamanders, such as Plethodon, move primarily by bodily undulations rather than on all fours. Further, their small size would significantly reduce the weight to be carried by their legs. On the other hand, larger aquatic salamanders that do not regenerate limbs may have lost that ability when they acquired the capacity to swim without using their limbs, the latter thereby losing the selective advantage of retaining regenerative competence. In the anurans, it is a curious fact that the limbs of tadpoles regenerate quite well, but lose this faculty after metamorphosis--at a time when they become most useful. Again, this paradox can be explained by the need for frog legs to support the body on land, a condition that would be difficult to reconcile with regeneration of normal replacements. Larval limbs, however, are essentially non-functional, or at least not weight-beating. Their regeneration enables the animal to emerge from metamorphosis with its legs intact. Xenopus laevis, the South African clawed toad, may be an exception that proves this rule. Adults retain the ability to regrow amputated legs, albeit abnormally so, but they also remain lifelong inhabitants of an aquatic environment. If this principle is applied to the higher vertebrates, why is it that the wings of birds or the flukes and flippers of cetaceans and pinnipeds do not regenerate as nonweight-bearing appendages? Such structures tend to be indispensable to the animals' locomotion, and as such are vitally essential. Even if they could regenerate, it is doubtful that the animals would survive long enough to complete their regrowth. Although cetaceans give birth at sea, seals, sea lions, and walruses all return to the land for mating and parturition, during which episodes their flippers are quite necessary for support and locomotion. The wings of birds are, of course, vital necessities, but one must wonder why (or if) those of flightless birds do not regenerate. Could it have to do with the warm blooded condition? Or are they just not necessary enough? Whether or not an appendage is weight-bearing actually relates to the question of mechanical contact, which need not necessarily be with the ground. In the male teleost, Gambusia, for example, part of the anal fin is modified as an intromittent organ, the gonopodium. While other fins regenerate, the specialized gonopodium does not (Turner, 1947), perhaps owing to its vulnerability to abrasian during mating. Deer antlers are also vulnerable. Though not weight-bearing, they are subject to the injuries of contact sport. The consequences of such mutilations are avoided, however, by two conditions. One is the behavioral suppression of aggressive tendencies while the growing antlers are in velvet. The other is the natural demise of the fully mature bony antler after the velvet has been shed. Tines may be broken off in rut, but whole new antlers are regenerated the next year. Horns, on the other hand, are sufficiently rugged to withstand epigamic combat because their growth zones are proximal, not on the exposed ends, and because active growth in seasonal breeders is held in abeyance while the animals are in rut. Homeothermy has made possible many advances without which life as we experience it could never have evolved. But it has also militated against regeneration. Being warm blooded requires a heightened metabolic rate, which can be sustained only by frequent nutrition. Unlike cold blooded vertebrates, which can survive months of

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starvation if necessary, warm blooded ones will die in days or weeks, depending on their size. Thus, a mammal crippled by the loss of a leg would be unable to catch its prey if it were carnivorous, or to escape its predators if it were herbivorous. It could hardly live long enough to regenerate a new leg. Under such hopeless conditions, there would be no selective pressure to preserve its regenerative capacity. Further, the warm blooded environment constitutes an excellent culture medium for the proliferation of pathogens. Open wounds are inconveniently susceptible to infections. This may explain why birds and mammals have evolved such efficient means of wound healing. The centripetal migration of epidermis (Stenn & Malhatra, 1992) is augmented by a conspicuous wound contraction (Rudolph et al., 1992). Together, they seal the raw surface with all deliberate haste. Underneath, the granulation tissue is the site of scar formation. Scars are regenerated dermis. Like the full thickness skin pulled inward over the wound by contraction, scars constitute a thick barrier of collagenous connective tissue interposed between the wound epidermis and the underlying mesodermal tissues. It is the latter, in lower vertebrates, from which blastema cells arise to congregate beneath the apical epidermal cap. Unless inductive interactions can take place between these epidermal and mesodermal tissues, blastema formation fails to occur. Therefore, it is generally believed that precocious scar formation in warm blooded vertebrates may act as a blockade to inductions requisite for the production of a blastema and the regenerate into which it would otherwise have developed. The loss of regeneration, for whatever proximate and ultimate reasons, is not necessarily an all-or-nothing phenomenon. In some forms, most notably the postmetamorphic anurans, varying degrees of regeneration are represented which, according to Scadding (1981), "lie along a continuum". Such examples of hypomorphic regeneration as described by Beetschen (1952), Dent (1962), Goode (1967), Michael & Al Sammak (1970), Scadding (1981), Richards et al. (1975), and Thornton & Shields 0945) are probably expressions of regrowth in the process of being phased out. The decline is both ontogenetic and phylogenetic, and in some cases (e.g. Xenopus laevis) would seem attributable to a deficiency in producing an adequate blastema (Komala, 1957; Skowron & Komala, 1957; Korneluk & Liversage, 1984).

6. Proximate Explanations There would seem to be alternative pathways open to an amputated stump. It could develop either a scar or a blastema, but not both. In lower vertebrates, whose limbs regenerate so well, no scar forms in the process of wound healing on an amputated stump, which may be why a blastema forms instead. The question arises, would a mammalian stump produce a blastema if scar formation were somehow prevented? It is conceivable that other factors (e.g. adequate innervation) might be involved too, but Occam's rule, the concepts ought not to be multiplied beyond necessity, would dictate that the simplest hypothesis be explored first before additional complications are factored into the formula. Thus, the most optimistic interpretation is that blastema formation may be automatic unless precluded by a scar.

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It makes sense that vitally essential appendages like legs and wings ought not to regenerate in warm blooded vertebrates because such animals would perish long before they could complete regeneration. The obverse of this argument is that appendages not vitally necessary for survival might be able to grow back. Deer antlers are a case in point (Goss, 1983). Their loss is not life-threatening, yet they serve a useful function in sexual selection between competing males. How antlers evolved in the first place, as they did about 30 million years ago in the Miocene, is open to conjecture. Nevertheless, these unique zoological curiosities are cast off each year and replaced by new ones which attain their full lengths in only a few months before turning into solid bone and shedding their velvet integument in time for the mating season. The existence of these remarkable cephalic appendages proves that there is nothing about being a mammal p e r se that renders regeneration impossible. Mammals can regenerate in certain exceptional circumstances (e.g. rabbit ears, children's finger tips), so the question is why some structures can regrow while others cannot (Goss, 1987). Comparisons between the ears of rabbits (which fill in holes 1 cm in diameter) and those of dogs and sheep (which do not) have yielded evidence that whether a blastema or a scar is formed on the cut surface depends on how the wound is healed (Goss & Grimes, 1972, 1975). In non-regenerative ears, the inner and outer epidermis migrates across the margins of the hole during the first week, whereupon scar tissue develops to complete the continuity of the interrupted dermis on either side. In regenerative ears, however, rather conspicuous epidermal downgrowths, present between 5 and 12 days postoperatively, form at the cut edges of the dermis. These long intrusions of epidermis, which may penetrate a millimeter or so beneath the surface, are in precisely the right place at exactly the right time to constitute a barrier to the regeneration of a scar from the dermis. In the absence of a scar, a mass of cells, comparable to a blastema in other systems, forms off the end of the interrupted cartilaginous sheet. Subsequent chondrogenesis reconstitutes the auricular cartilage across the gap. The epidermal downgrowths become flattened out as the regenerate elongates. Circumstantial evidence is insufficient to prove that regeneration in this case is permitted because scar formation is prevented by epidermal downgrowths, but the hypothesis would be worth putting to the experimental test. No such histological configurations have been observed in the regenerating deer antler. Here, the mesodermal source of the antler bud appears to be two-fold. One derives from within the interstices between the bony trabeculae of the pedicle just before the old antler is detached by osteoclastic erosion. The other appears to invade the raw surface of the pedicle immediately after loss of the dead antler by centripetal migration from the surrounding skin. Whether the latter tissues are dermal, periosteal, or from other non-descript connective tissue, is difficult to ascertain. Nonetheless, scar tissue is conspicuous by its absence from the pedicle wound from which the antler regenerates. It has been speculated that the very tissue which elsewhere in the body would give rise to a scar might in this location transform into an antler bud (or blastema). If this interpretation were valid, then the deer antler would be an exaggerated modification of the scar itself. The foregoing studies of mammalian regeneration suggest that there is more than one way for lost structures to replace themselves. Similarly, there are undoubtedly

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multifarious proximate mechanisms by which mammalian and avian appendages might be prevented from regenerating. Assuming that even in these cases there remains a residual regeneration phenotype unexpressed because of some blockade, the task is to determine what the nature of these blockades might be in order to remove or bypass them. Weismann was right when he related the power of regeneration to the liability to injury. Like other genotypes, regeneration can only be expressed when conditions permit. Melanogenesis depends upon exposure to ultraviolet radiation. The development of secondary sex characters cannot be expressed in the absence of sex hormones. There is no aggressive behavior without an antagonist. So it is that the potential for regeneration remains latent unless amputation triggers its onset. Indeed, the only way to predict whether or not an appendage can regenerate at all is to cut it off and observe the results. This is also how natural selection works. If a structure otherwise capable of regeneration is never amputated, or its loss is inevitably fatal, there is no way its capacity for regrowth can be protected against the onslaughts of fortuitous mutations deleterious to regeneration. Only by being subjected frequently enough to the acid test of natural selection can a structure or process be preserved in the course of evolution. This tentative conclusion, however, is predicated on the assumption that antiregenerative mutations will naturally supervene if an appendage's capacity for regeneration is too infrequently exercised. Are there any examples of the persistence of other unexpressed capacities over long periods of evolution? There comes to mind the interesting example of dental amelogenesis exhibited by avian ectoderm when appropriately induced by mouse dental mesoderm in the chorioallantoic membrane (Kollar & Fisher, 1980). If the chick ectoderm in these experiments was not contaminated by mouse epidermis, then even after the many millions of years since birds ceased to develop teeth, their tissues have nonetheless retained the latent capacity to do so. Other examples are certain insects in which males are unknown in parthenogenetic species. Yet the females still retain their seminal receptacles. Nature would seem to be sufficiently conservative to preserve unused capacities (as well as structures) for prolonged stretches of evolutionary history. This suggests that a latent capacity constitutes little or no drain on metabolic economy, at least not enough to be susceptible to negative selection. This principle also ought to apply to regenerative capacities even when no longer needed. They may be unexpressed when amputations are negligibly infrequent, or when animals cannot survive the loss of vital appendages, or when precluded by scars or insufficient innervation, but in all likelihood the capacity to regenerate has not been extinguished altogether. "Whether regeneration is induced spontaneously or by an investigator", wrote Reichman (1984), "there is strong evidence that the basic ability is present even though the appropriate trigger mechanism may not be".

7. The Utilitarian Imperative It is a curious fact of regeneration that it has evolved a dependence on physiological agencies closely bound up with the normal function of the appendage to be replaced.

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This utilitarian imperative ensures that the regenerate will be functional. It is expressed in a variety of ways, and would seem to have been selected for its role in avoiding the wasteful expenditure of resources in the regeneration of useless structures. Neurotrophic influences are by far the most important factors in stimulating appendage regeneration (Singer & G~raudie, 1991). This makes sense because the function of the body's appendages are almost by definition dependent upon their innervations. Thus, an adequate threshold of nerves is necessary not only for the regeneration of amputated amphibian limbs, but also for the regrowth of teleost fins (Goss & Stagg, 1957) and taste barbels (Kamrin & Singer, 1953). However, they are not required for jaw regeneration in the salamander (Finch, 1969), which depends upon the mandible instead. An intact spinal cord is necessary for the regeneration of tails in urodeles (Holtzer, 1956) and lizards (Simpson, 1970), but not in tadpoles, in which tails fail to regrow without the notochord (Morgan & Davis, 1902). Deer antlers, though richly innervated while in velvet, can grow even when denervated (Wislocki & Singer, 1946). As secondary sex characters, their production is regulated by seasonal variation in testosterone levels. Among the invertebrates, annelid worms fail to regenerate anteriorly if the ventral nerve cord has been removed (Avel, 1961). Their posterior regeneration, however, depends upon the presence of an intestine (Boilly, 1969). In flatworms, eyes do not regenerate without a brain (Lender, 1952). In arthropods, regeneration does not occur unless the molting hormone promotes ecdysis (Passano & Jyssum, 1963). Compensatory growth of internal organs and tissues also reflects the importance of functional demands (Goss, 1978). Endocrine glands do not enlarge after partial ablation unless appropriate trophic hormone levels are high enough, or the electrolytes they are responsible for are at certain concentrations in the blood. Red cell production is sensitive to erythropoietin, produced in proportion to oxygen demands. The heart reacts to hypertension, the skeletal muscles to tension and exercise, the prostate to testosterone, taste buds to sensory nerves, and salivary glands to autonomic innervation. Again and again, growth is linked to function, which assures that regeneration in all of its many manifestations will be adaptively advantageous. It is difficult to say which came first, form or function. Embryologically, however, organs and tissues tend to differentiate before they are physiologically competent. Limb buds are formed prior to becoming innervated. Tail bud development precedes spinal cord differentiation. In the regeneration or compensatory growth of adult structures, however, the sequence is inverted. Growth does not commence until it is stimulated by the specific physiological influences without which function could not be assured. This reverse pattern suggests that in regeneration appropriate physiological dependencies have evolved secondarily. Such adaptations have obvious survival values, and were therefore favored by natural selection. The selection is not perfect, however. Denervated limbs, for example, do not regenerate because they are paralyzed, but because they lack nerves. If limbs are rendered useless by severing the spinal cord while leaving the spinal neurons intact, regeneration proceeds anyway. Alternatively, it is possible to anesthetize salamander larvae for prolonged periods, during which time their amputated limbs can still

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regenerate (Hui & Smith, 1970). Finally, by removing segments of the spinal cord and the associated ganglia from embryos, nerveless (aneurogenic) limbs will develop which can nevertheless regenerate after amputation (Yntema, 1959). Clearly, natural selection has blindly allowed the regeneration of limbs whenever the nerve supply surpassed a critical threshold, regardless of whether or not such nerves were functional. The results have been adaptive under natural conditions (but not in the experimental laboratories never anticipated by evolution).

8. Autotomy If regeneration is in fact not adaptive, there can be little doubt that autotomy is. This is the phenomenon by which structures are actively cast off, and its many expressions among animals fulfills the criteria by which adaptation by natural selection is defined. Autotomy is the self-amputation of appendages triggered by external agencies, usually aggressive in nature. Actually a pre-adaptation that anticipates offensive encounters, it has evolved as a mechanism that enables potential prey to sacrifice appendages and thereby to escape their predators. In many cases, the latter are distracted by the wriggling of the grasped structure which simulates the writhing of an apprehended victim. Autotomy occurs in many different animals. Among the invertebrates, crustaceans are notorious for their propensity to surrender their legs or chelae. The limbs of centipedes and arachnids are often readily detached when grasped. Starfish tend to autotomize injured arms near the base, and octopuses may do likewise at any level if they are inextricably caught (Lange, 1920). Among cephalopods, the male octopus and argonaut possess an arm modified for mating, the hectocotylus. This appendage breaks off in the throes of copulation, continuing its autonomous existence within the female's mantle (Lane, 1960). Autotomy in the vertebrates is limited to tails. Some urodele amphibians (e.g. Plethodon) may disconnect parts of their tails at will, and lizards are famous for their ability to drop their tails and run. Mechanisms of autotomy have been investigated in crustaceans and lizards. Typically, there is a preformed cleavage plane where breakage occurs. In the crab, it is between the basis and ischium at the proximal end of the leg (Bliss, 1960). In reptilian tails, it is through the middle of each vertebra (Pratt, 1946). Either way, the nervous stimulation of being grasped or pinched initiates a reflex reaction resulting in the forceful contraction of muscles across the level of detachment and subsequent separation of the appendage from its stump. The muscular convulsions may persist distally, thus diverting the attackers' attention. Excessive bleeding is controlled by a septum valve across the crustacean appendage, or by vasoconstriction in lizard tails. Autotomy has clearly evolved independently here and there as an adaptation to break loose when one's appendages have been trapped. It would be expected to have been selected among animals vulnerable to attack. Thus it is most prevalent in smaller species living in competitive habitats, species which depend on escape mechanisms to survive and reproduce. Autotomy is usually, but not always, accompanied by regeneration. For example, if a rat is suspended by its tail it will spin violently enough to slip out of its tail skin, which comes of as an empty sleeve. The denuded end of

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the tail dies, of course, and is not regenerated. The segmental nature of the skin, and its loose attachment to underlying tissues, would seem to be an adaptation favoring self-preservation not unlike the autotomy of lizard tails. In the vast majority of cases, autotomy is followed by regeneration. Which came first is a matter of conjecture, but it is probably safe to assume that the autotomy mechanism has been superimposed on the pre-existing ability to regenerate. This combination re-equips the animal with the capacity for subsequent episodes of autotomy and regeneration, each round saving its life and enhancing the chances for procreation. The advantages of this are so obvious that one wonders why natural selection has not given rise to similar adaptations in other regenerating appendages, such as fish fins and salamander legs.

9. Prospects Although autotomy and neurotrophic effects are clearly adaptive, regeneration itself is not. Available evidence suggests that it is probably a primeval attribute of metazoans related to embryonic development which it so closely resembles. This interpretation explains the basic similarities between different regenerative phenomena, consistent with their monophyletic derivation. On the other hand, such losses of regenerative abilities as have occurred heterogeneously during the course of evolution would appear to be polyphyletic, each linked pleiotropically to adaptations more useful than regeneration. While it is encouraging to suspect that regenerative potentials may lie latent in non-regenerative appendages, it is daunting to realize that wherever regeneration has been lost there are probably different mechanisms responsible for the inhibition. If it is a goal of regeneration research to induce regrowth where it does not naturally occur, then it will be important to pinpoint in each case what the most plausible blockade to regeneration may be. Comparisons of regenerative and nonregenerative structures, especially when they are closely related, can reveal histological differences between them, differences that might be proximately responsible for why one appendage regenerates and another does not. This strategy could then serve as a basis for designing experiments to circumvent impediments to the onset of regeneration. REFERENCES ALLEN, G. E. (1978). Thomas Hunt Morgan. The Man and His Science. Princeton, NJ: Princeton University Press. AVEL, M. (1961). L'influence du syst/:me nerveux sur la rrgrn&ation chez les urodrles et les oligochrtes. Bull. Soc. zool. Fr. 86, 464483. BARR, H. J. (1964). Regeneration and natural selection. Am. Nat. 98, 183-186. BEETSCHEN, J.-C. (1952). Extension et limites du pouvoir rrgrnrrateur des membres aprrs la mrtamorphose chez Xenopus laevis Daudin. Bull. biol. Fr. Belg. 86, 88-100. BLISS, D. E. (1960). Autotomy and regeneration. In: The Physiology of Crustacea. L Metabolism and Growth (Waterman, T. H., ed.) pp. 561-589. New York: Academic Press. BOILLY, B. (1969). Sur la nature du tissu a l'origine de 1'rpithrlium intestinal au tours de la rrg~n&ation chez Syllis arnica Quatrefages (Annrlide Polychrte). l~tude exprrimentale. Annls d'Embryol. Morphogen~se. 2, 343-354.

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BONNET, C. (1745). Traitib d'Insectologie, Volume 2. Paris: Duvand Libraire. BRODY, S. (1945). Bioenergetics and Growth. Princeton, N J: Van Nostrand-Reinhold. CARROLL, R. L. & BAreD, D. (1968). The carboniferous amphibian Tuditanus [Eosauravus] and the distinction between microsaurs and reptiles. Am. Mus. Nooit. 2,337, 1-50. COATES, M. (1991). New palaeontological contributions to limb ontogeny and phylogeny. In: Deoelopmental Patterning of the Vertebrate Limb (Hinchcliffe, J. R., Hurle, J. M. & Summerbell, D., eds) pp. 325-337. New York: Plenum Press. COMFORT, A. (1964). Ageing. The Biology of Senescence. New York: Holt, Rinehart & Winston. DENT, J. N. (1962). Limb regeneration in larvae and metamorphosing individuals of the South African clawed toad. J. Morph. 110, 61-77. ELDER, D. (1979). Why is regenerative capacity restricted in higher organisms? J. theor. Biol. 81,563-568. FINCH, R. A. (1969). The influence of the nerve on lower jaw regeneration in the adult newt, Triturus oiridescens. J. Morph. 129, 401--414. GOODE, R. P. (1967). The regeneration of limbs in adult anurans. J. EmbryoL exp. Morph. 18, 259-267. Goss, R. J. (1978). The Physiology of Growth. New York: Academic Press. Goss, R. J. (1980). Prospects for regeneration in man. Clin. Orthop. 151, 270-282. Goss, R. J. (1983). Deer Antlers: Regeneration, Function, and Eoolution. New York: Academic Press. Goss, R. J. (1987). Why mammals don't regenerate---or do they? News physiol. Sci. 2, 112-115. Goss, R. J. & GRIMES, L. N. (1972). Tissue interactions in the regeneration of rabbit ear holes. Am. Zool. 12, 151-157. Goss, R. J. & GRIMES, L. N. (1975). Epidermal downgrowths in regenerating rabbit ear holes. J. Morph. 146, 533-542. Goss, R. J. & STAGG, M. W. (1957). The regeneration of fins and fin rays in Fundulus heteroclitus. J. exp. Zool. 136, 487-508. HOLTZER, S. (1956). The inductive activity of the spinal cord in urodele tail regeneration. J. Morph. 99, I--40. Hul, F. & SMITH, A. (1970). Regeneration of the amputated amphibian limb: retardation by hemicholinium-3. Science 170, 1313-1314. HUXLEY, J. S. (I 943). Evolution, The Modern Synthesis. New York: Harper. JACOB, F. (1977). Evolution and tinkering. Science 196, 1161-1166. JAMISON, J. (1964). Regeneration subsequent to intervertebral amputation in lizards. Herpetologica. 20, 145-149. KAMRIN, R. P. & SINGER, M. (1953). Influence of sensory neurons isolated from central nervous system on maintenance of taste buds and regeneration of barbels in the catfish, (Ameiurus) nebulosus. Am. J. Physiol. 174, 146-148. KING, H. D. (1898). Regeneration in Asterias vulgaris. Arch. EntwMech. Org. 7, 351-363. KING, H. D. (1900). Further studies on regeneration in Asterias oulgaris. Arch. EntwMech. Org. 9, 724-737. KOLLAR, E. & FISHER, C. (1980). Tooth inductions in chick epithelium: expression of quiescent genes for enamel synthesis. Science 207, 993-995. KOMALA, Z. (1957). Comparative investigations on the course of ontogenesis and regeneration of the limbs in Xenopus laeois tadpoles in various stages of development. Folia bioL, Krakow 5, 1-51. KORNELUK, R. G. & LIVERSAGE,R. m. (I 984). Tissue regeneration in the amputated forelimb of Xenopus laeois froglets. Can. J. Zool. 62, 2383-2391. KORSCHELT, E. (1927). Regeneration und Transplantation. Berlin: Gebriider Borntraeger. LANE, F. W. (1960). Kingdom of the Octopus. The Life History of the Cephalopoda. New York: Sheridan House. LANGE, M. M. (1920). On the regeneration and finer structure of the arms of the cephalopods. J. exp. ZooL 31, 1-57. LENDER, T. (1952). Le rrle inducteur du cerveau dans la rrgSnrration des yeux d'une Planaire d'eau douce. Bull. bioL Ft. Belg. 86, 140-215. MARKELOVA, I. V. (1952). Regeneration of nipples in rabbits. Bull. exp. Biol. Med. 33(6), 58-62. MICHAEL, M. 1. & AL SAMMAK, A. J. (1970). Regeneration of limbs in adult Rana ridibunda ridibunda Pallas. Experientia. 26, 920-921. MORGAN, T. H. (1898). Regeneration and liability to injury. Zool. Bull. !, 287-300. MORGAN, T. H. (1901). Regeneration. New York: The Macmillan Co. MORGAN, T. H. (1932). The Scientific Basis of Evolution. New York: W. W. Norton. MORGAN, T. H. & DAVIS, S. E. (1902). The internal factors in the regeneration of the tail of the tadpole. Arch. EntwMech. Org. 15, 314-318. NEEDHAM, A. E. (1961a). Adaptive value of regenerative ability. Nature, Lond. 191, 720-721.

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NEEDHAM, A. E. (1961b). Evolution of regeneration and its possible bearing on philosophy. Nature, Lond. 192, 1255-1256. PARKER, W. S. (1972). Aspects of the ecology of a Sonoran Desert population of the western banded gecko, Coleonyx variegatus (Sauria, Eublepharinae). Am. MidL Nat. 88, 209-224. PASSANO, L. M. & JYSSUM, S. (1963). The role of the Y-organ in crab proecdysis and limb regeneration. Cutup. Biochem. Physiol. 9, 195-213. PRATT, C. W. M. (1946). The plane of fracture of the caudal vertebrae of certain Lacertilians. J. Anat. Lond. 80, 184-188. RI~AUMOR, R. A. DE. (1742). Mdmoires Pour Seruir d l'historie des Insects, Volume 6. Paris: Imprimerie Royale. REIOtMAN, O. J. (1984). Evolution of regeneration capabilities. Am. Nat. 123, 752-763. R1CHARDS, C. M., CARLSON, B. M. & ROGERS, S. L. (1975). Regeneration of digits and forelimbs in the Kenyan reed frog Hyperolius oiridiflaousferniquei. J. Morph. 146, 431--446. RUDOLPH, R., BERG, J. V. X, EHRLICH, H. P. (1992). Wound contraction and scar contracture. In: Wound Healing. Biochemical and Clinical Aspects (Cohen, I. K., Diegelmann, R. F. & Lindblad, W. J., eds) pp. 96-114. Philadelphia, PA: W. B. Saunders. SCADDmG, S. R. (1977). Phylogenic distribution of limb regeneration capacity in adult Amphibia. J. exp. Zool. 202, 57-68. SCADDING, S. (1978). Limb regeneration in Amphiuma tridactylum (Amphibia, Urodela). Can. J. ZooL 56, 2327-2332. St:ADDING, S. R. (1981). Limb regeneration in adult amphibia. Can. J. Zool. 59, 34-46. SIMPSON, S. B., JR. (1970). Studies on regeneration of the lizard's tail. Am. Zool. 10, 157-165. StaGER, M. & Gt~RAUDm, J. (I 991 ). The neurotrophic phenomenon : its history during limb regeneration in the newt. In : A History of Regeneration Research. Milestones in the Eoolution of a Science (Dinsmore, C. E., ed.) pp. 101-112. Cambridge: Cambridge University Press. SKOWRON, S. & KOMALA, Z. (1957). Limb regeneration in post-metamorphic Xenopus laevis. Folia biol., Krakow. 5, 53-72. S'rEr,rN, K. S. & MALHATRA, R. (1992). Epithelialization. In: Wound Healing. Biochemical and Clinical Aspects (Cohen, I. K., Diegelmann, R. F. & Lindblad, W. J., eds) pp. 115-127. Philadelphia, PA: W. B. Saunders. S'rOCOM, D. L. (1991). Limb regeneration: a call to arms (and legs). Cell 67, 5-8. THORNTON, C. S. • SHIELDS, T. W. (1945). Five cases of atypical regeneration in the adult frog. Copeia. 1, 40-42. TURNER, C. L. (1947). The rate of morphogenesis and regeneration of the gonopodium in normal and castrated males of Gambusia affinis. J. exp. ZooL 106, 125-143. VITT, L., CONGDON, J. & DICKSON, N. (1977). Adaptive strategies and energetics of tail autotomy in lizards. Ecology 58, 326-337. WAGNER, G. P. & MISOF, B. Y. (1992). Evolutionary modification of regenerative capability in vertebrates: a comparative study on teleost pectoral fin regeneration. J. exp. Zool. 261, 62-78. WEISMANN,A, (I 893). The Germ-Plasm. A Theory of Heredity (Translated by Parker, W. N. & R6nnfeldt, H.) New York: Charles Scribner's Sons. WEISMANN, A. (1899). Regeneration: facts and interpretations. Nat. Sci. 14, 305-328. WERNER,Y. L. (1968). Regeneration frequencies in geckos of two ecological types (Reptilia: Gekkonidae). Vie Milieu. Ser. C. 19, 199-222. WISLOCKI,G. n. ~,~SINGER,M. (1946). The occurrence and function of nerves in the growing antlers of deer. J. cutup. NeuroL 85, 1-19. YNTEMA, C. L. (1959). Regeneration in sparsely innervated and aneurogenic forelimbs of Amblystoma larvae. J. exp. ZooL 140, 101-124.